In an effort to identify new G-protein coupled receptors of the EDG (endothelial differentiation gene)-family a novel member of the EDG-family of G-protein coupled receptors, Human EDG8, was identified. The full-length clone was isolated and studies on chromosomal mapping, tissue expression and identification as a functional cellular receptor for sphingosine 1-phosphate were performed. Taken together, the data provide compelling evidence that EDG8 is the fifth receptor for sphingosine 1-phosphate, exclusively expressed in peripheral tissues, its presence in endothelial cells being responsible for the broad tissue distribution.
The lysolipid phosphate mediators lysophosphatidic acid (LPA) and sphingosin 1-phosphate (S1P) have attracted increasing attention as modulators of a variety of important biological functions (Moolenaar et al., Current Opinion in Cell Biol 9:168, 1997; Morris, Trends Pharmacol Sci 20:393, 1999; Lynch and Im, Trends in Pharmacol Sci 20:473, 1999) and their list of biological activities is continuously growing.
Among the biological responses to LPA is platelet aggregation (Jalink et al., Biochem Biophys Acta 1198:185, 1994; Siess et al., PNAS USA 96:6931, 1999; Gueguen et al., Biochemistry 38: 8440, 1999), smooth muscle contraction (Tokumura et al., Arch Int Pharmacodyn Ther 245:74, 1980), in vivo vasoactive effects (Tokumura et al., Res Comm Mol Pathol Pharmacol 90:96, 1995), chemotaxis (Jalink et al., PNAS USA 90:1857, 1993), expression of adhesion molecules (Lee et al., J Biol Chem 273:22105, 1998; Rizza et al., Laboratory Investigation 79:1227, 1999), increased tight junction permeability of endothelial cells (Schulze et al., J Neurochem 68:991, 1997), induction of stress fibers (Gohla et al., J Biol Chem 274:17901, 1998) and many others (for review see Moolenaar et al., Current Opinion in Cell Biol 9:168, 1997). The biochemical signalling events that mediate the cellular effects of LPA include stimulation of phospholipases, mobilization of intracellular Ca2+, inhibition of adenylyl cyclase, activation of phosphatidylinositol 3-kinase, activation of the Ras-Raf-MAP kinase cascade and stimulation of Rho-GTPases (Moolenaar et al., Current Opinion in Cell Biol 9:168, 1997).
S1P, in particular, is implicated in cell proliferation, modulation of cell motility (reviewed in Hla et al., Biochem Pharm 58:201, 1999) induction/suppression of apoptosis (Hisano et al., Blood 93:4293, 1999; Xia et al., J Biol Chem 274:34499, 1999), angiogenesis (Lee et al., Cell 99:301, 1999), tumor invasiveness (Sadahira et al., PNAS USA 89:9686, 1992), platelet activation (Gueguen et al., Biochemistry 38:8440, 1999) and neurite retraction (Postma et al., EMBO J 15:2388, 1996). Cellular signalling by S1P involves activation of PLCβ and subsequent intracellular Ca2+ release (van Koppen et al., J Biol Chem 271:2082, 1996; Meyer zu Heringdorf et al., Naunyn-Schmiedeberg's Arch Pharmacol 354:397, 1997; Yatomi et al., J Biol Chem 272:5291, 1997a; Noh et al., J Cell Physiol 176:412, 1998; Ancellin and Hla, J Biol Chem 274:18997, 1999), activation of MAP-kinases (Wu et al., J Biol Chem 270:11484, 1995; Lee et al., J Biol Chem 271:11272, 1996; An et al., J Biol Chem 275:288, 2000), activation of inward rectifying K+-channels (van Koppen et al., J Biol Chem 271:2082, 1996; Bünemann et al., EMBO J 15:5527, 1996) and inhibition and/or activation of adenylyl cyclase (Lee et al., J Biol Chem 271:11272, 1996).
Both, LPA and S1P are recognized to signal cells through a set of G-protein coupled receptors (GPCRs) known as EDG (endothelial differentiation gene)-receptors. The EDG-family of GPCRs currently comprises seven human members (EDG1-7) that fall into two major groups depending on their preference for the activating lipid-ligand: EDG1, 3, 5 and 6 preferentially interact with S1P (Yatomi et al., J Biochem (Tokyo) 12:969, 1997; Lee et al., Science 279:1552, 1998; Lee et al., J Biol Chem 273:22105, 1998; Ancellin and Hla, J Biol Chem 274:18997, 1999; Yamazaki et al., Biochem Biophys Res Commun 268:583, 2000; Van Brocklyn et al., Blood 95:2624, 2000), EDG2, 4 and 7 preferentially interact with LPA (An et al., J Biol Chem 273:7906, 1998; Im et al., Mol Pharmacol 57:753, 2000).
The assignment of specific biological functions to certain receptor subtypes is hampered by the fact that EDG receptors are expressed in an overlapping fashion (Rizza et al., Laboratory Investigation 79:1227, 1999; Lee et al., Cell 99:301, 1999), they activate multiple and in part redundant signal transduction pathways (Lee et al., J Biol Chem 271:11272, 1996; Ancellin and Hla, J Biol Chem 274:18997, 1999; Kon et al., J Biol Chem 274:23940, 1999; An et al., J Biol Chem 275:288, 2000), the selectivity for their activating ligands is not absolute (Lee et al., J Biol Chem 273:22105, 1998), and medicinal chemistry is only poorly developed in that specific antagonists for dissecting the pharmacology of the individual subtypes are not available yet.
An important step to shed more light on the biological role of the individual receptor subtypes would be to identify the complete set of receptors that respond to the phospholipid mediators S1P and LPA.